Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Polypeptide targeting to mitochondria

a technology of polypeptides and mitochondria, applied in the direction of fusion polypeptides, peptide/protein ingredients, depsipeptides, etc., can solve the problem of insufficient use of mts

Active Publication Date: 2009-07-23
UK RES & INNOVATION LTD
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0026]Where the effector molecule is a restriction enzyme, the unique site preferably does not occur in wild type mitochondria. In this way, the genomes of mitochondria harbouring a mutation can be selectively inactivated.

Problems solved by technology

It appears that the use of an MTS is not sufficient for the delivery of DNA-binding polypeptides heterologous to the mitochondrial environment.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Polypeptide targeting to mitochondria
  • Polypeptide targeting to mitochondria
  • Polypeptide targeting to mitochondria

Examples

Experimental program
Comparison scheme
Effect test

example 1

Strategies for Delivering ZFPs to Mitochondria

[0235]Zinc fingers are predominantly DNA-binding proteins, evolutionary adapted to operate in the nucleus. Even in the absence of nuclear localisation signals (NLS) they often localise in the nucleus (Papworth, M., Kolasinska, P. & Minczuk, M. Gene in press (2005)). To use designer ZFP to manipulate mtDNA, they have to be both effectively targeted to mitochondria but also at the same time to be absent from the nucleus to avoid binding to nuclear DNA which could be toxic (specific examples discussed in Papworth et al. Proc Natl Acad Sci USA 100, 1621-6 (2003)). The majority of mitochondrial proteins are encoded by nuclear genes and imported from the cytoplasm with an aid of a cleavable N-terminal mitochondrial targeting sequence (MTS). The MTSs vary greatly in length and composition and appear to be individually tailored to different proteins (Pfanner, N. & Geissler, A. Nat Rev Mol Cell Biol 2, 339-49 (2001)). Fusing an MTS to N-termini c...

example 2

Construction of a Mitochondria-Targeted ZFP that Binds a Particular mtDNA Sequence

[0241]The next step was to generate ZFPs that bound selectively to particular mtDNA sequences. To this end we created a mitochondrially targeted 3-finger protein F-ZFPNARP designed to bind a 9 bp sequence GCCCGGGCC in mtDNA (FIG. 2A); the bold G is at position 8993 in mtDNA and indicates a mutation responsible for the mitochondrial diseases Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP) and maternally-inherited Leigh syndrome (MILS). The wild type (wt) sequence has a T at this position. In vitro FZFPNARP specifically bound an oligonucleotide containing GCCCGGGCC, as assessed by a gel retardation assay (FIG. 2B), In contrast, oligonucleotides containing either GCCCTGGCC (wt) or GCCCCGGCC were not bound by F-ZFPNARP (FIG. 2B). Additionally, in vitro binding studies of ZFPs showed that the addition of NES did not affect DNA binding (data not shown). A control, F-ZFPcont, of the same si...

example 3

Targeting a Chimaeric ZFP-Methylase to a Particular mtDNA Sequence

[0246]The next step was to investigate whether F-ZFPNARP could direct a DNA modifying activity selectively to the mutant GCCCGGGCC mtDNA sequence. As a DNA modifying activity we chose the catalytic domain of the human DNMT3a DNA methyltransferase. This enzyme predominately methylates cytosines in CpG sites to 5-methylcytosine (m5C), using S-adenosylmethionine (SAM) as a substrate. Chimaeric proteins comprising Zif268-derived ZFPs and the catalytic DNA methyltransferase domains have previously been shown to catalyse sequence specific DNA methylation in vitro (Xu, G. L. & Bestor, T. H. Nat Genet 17, 376-8 (1997); McNamara, A. R., Hurd, P. J., Smith, A. E. & Ford, K. G. Nucleic Acids Res 30, 3818-30 (2002)) and in vivo (Carvin, C. D., Parr, R. D. & Kladde, M. P. Nucleic Acids Res 31, 6493-501 (2003)). Endogenous cytosine methylation in human mtDNA is very limited (Shmookler Reis, R. J. & Goldstein, S. J Biol Chem 258, 90...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Gene expression profileaaaaaaaaaa
Login to View More

Abstract

Methods for delivering non-mitochondrial proteins to mitochondria are provided. Also provided are nucleic acid constructs comprising a coding sequence encoding a DNA-binding polypeptide, fused to a mitochondrial targeting sequence (MTS) and a nuclear export signal (NES), and the encoded proteins. The construct successfully delivers DNA binding proteins to the mitochondrion. A chimeric methylase based on the above construct is successfully delivered to mitochondria, resulting in modification of mtDNA.

Description

[0001]The present invention relates to the targeting of polypeptides to the mitochondrion. In particular, the invention relates to the mitochondrial targeting of polypeptides which are heterologous to the mitochondrion, such as polypeptides with very strong nuclear localisation which are resistant to delivery to the mitochondrion. The invention moreover relates to methods for modifying mitochondrial DNA using polypeptides delivered to the mitochondrion.BACKGROUND[0002]Mitochondria are cellular organelles found in eukarytotic cells that play a central role in energy metabolism, apoptosis and ageing. Mitochondria contain a distinct mitochondrial genome, and human mitochondria contain 2 to 10 copies of their own DNA (mtDNA) of 16,569 bp, which encodes essential components of the oxidative phosphorylation machinery. This makes the mitochondrion to a certain extent independent of the nucleus, in that proteins are synthesised directly in the mitochondrion. Mitochondrial DNA resembles prok...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): A61K38/00C12N15/63C07H21/04C07K14/00C12N9/14A61K31/7088C12N5/00C12P21/06C12N15/00
CPCC07K14/47C07K2319/81C07K2319/07A61P1/04A61P21/04A61K38/1709C12Y306/03014C12N9/1007C12Y201/01072C12N9/14A61K38/45A61K38/46A61P25/16A61P25/28A61P27/00A61P27/16A61P43/00A61P9/00A61P9/10A61P3/10C07K2319/095
Inventor MINCZUK, MICHALPAPWORTH, MONIKA A.KLUG, AARON
Owner UK RES & INNOVATION LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com